Methods and vectors for site-specific recombination in plant cell plastids

ABSTRACT

Novel compositions and methods useful for genetic engineering of plant cells are provided. In particular, plastid constructs comprising recombining sites are provided. Such constructs find use in methods for site specific recombination in plant cell plastids. Plant cells and plants comprising the constructs described herein, as well as plant cells and plants produced by the methods of the present invention are of interest.

This application claims priority to U.S. provisional applications60/159,876 filed Oct. 15, 1999 and 60/225542 filed Aug. 16, 2000, hereinincorporated by reference in their entirety.

INTRODUCTION

1. Technical Field

This invention relates to the application of genetic engineeringtechniques to plants. More specifically, the invention relates toconstructs and methods for use of site specific recombination systemsfor the production of plastid transformed plants.

2. Background

Molecular biological techniques have enabled researchers to introducepieces of DNA from one organism to another organism. Such techniques,referred to as recombinant DNA technology, have positively impacted theareas of medicine and agriculture. Conventional cloning methods haveenabled the introduction of new pharmaceuticals and improved crops ofagricultural importance. As the need for the introduction of multiplepieces of DNA and larger fragments of DNA into various target hostsincreases, the need for novel cloning strategies increases accordingly.

The plastids of higher plants are an attractive target for geneticengineering. Plant plastids (chloroplasts, amyloplasts, elaioplasts,chromoplasts, etc.) are the major biosynthetic centers that in additionto photosynthesis are responsible for production of industriallyimportant compounds such as amino acids, complex carbohydrates, fattyacids, and pigments. Plastids are derived from a common precursor knownas a proplastid and thus the plastids present in a given plant speciesall have the same genetic content. Plant cells contain 500-10,000 copiesof a small 120-160 kilobase circular genome, each molecule of which hasa large (approximately 25 kb) inverted repeat. Thus, it is possible toengineer plant cells to contain up to 20,000 copies of a particular geneof interest, which potentially can result in very high levels of foreigngene expression.

Previous studies directed to stable transformation of plant chloroplastshave relied on homologous recombination to incorporate desired geneconstructs into plastids using a selectable marker for selection oftransplastomic plants. In this manner, transgenic plants homoplastic, ornear-homoplastic, for a recombinant DNA construct may be obtained.However, at present, methods for multiple rounds of plastidtransformation (for example for gene stacking) are restricted due to thelimited number of selectable markers described for plastidtransformation. Thus, there is a need in the art for constructs andmethods for performing multiple rounds of plastid transformation. Thisis done by removing the selectable marker gene after each round oftransformation by site-specific recombination.

SUMMARY OF THE INVENTION

By this invention, constructs and methods for genetic engineering ofplant cells to provide for site-specific recombination of foreign DNAsequences inserted into the plant plastid are provided.

In a first aspect of the present invention recombinant nucleic acidconstructs are provided that are useful for site-specific recombinationof nucleic acid sequences in a plant cell plastid. In particular,plastid constructs are provided that comprise at least one DNA sequence,and at least two recombining sites. Particularly preferred constructsare those that employ Lox recombining sites.

Another aspect of the present invention provides recombinant nucleicacid constructs having two DNA sequences with the recombining sitespositioned between the DNA sequences.

A further aspect of the present invention provides recombinant nucleicacid constructs having a DNA sequence positioned between the recombiningsites.

Another aspect of the present invention are recombinant nucleic acidconstructs comprising a transcription initiation region functional in aplant cell, an organelle targeting sequence, and a nucleic acid sequenceencoding recombinase. Such constructs are referred to herein asrecombinase constructs.

The recombinase constructs of the present invention provide forexpressing a recombinase in host plant cell tissues. Such constructsinclude plastid constructs and nuclear constructs. Nuclear constructsinclude those that provide for constitutive expression of therecombinase in all plant cells and those that provide for expressionpreferentially in particular plant tissues and/or at particulardevelopmental stages.

Also considered part of the present invention are the plants and plantcells comprising the constructs of the present invention.

Another aspect of the present invention is to provide methods fordirecting site-specific recombination in a host plant cell plastid. Inparticular, host plant cells are transformed or transfected with theplastid constructs having at least one DNA sequence and at least tworecombining sites. The method further comprises providing a recombinaseinto the plant cell.

In a further aspect, the present invention relates to methods ofretransforming a plant cell plastid using the same selectable marker.

Also considered in the present invention are the plant cells and plantsproduced by the methods described herein.

DESCRIPTION OF THE FIGURES

FIG. 1 provides the aadA plastid expression cassette positioned betweentwo loxP recombining sites. FIG. 1 also shows the loxP target sequencethat comprises two 13-base pair (bp) inverted repeats, and a central orcore 8-bp sequence referred to as the “spacer region.”

FIG. 2 provides a schematic diagram of the plastid construct pMON53117.

FIG. 3 provides a schematic diagram of the plastid construct pMON53119.

FIG. 4 provides a schematic diagram of the nuclear construct pMON49602.

FIG. 5 provides a schematic diagram of the nuclear construct pMON53147.

FIG. 6 provides a schematic diagram of the nuclear construct pMON49608.

FIG. 7 shows an alternate excision in clone Nt-Act2-53119-38.

FIG. 8 shows an alternate excision in clone Nt-35S-53119-2.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, constructs and methods areprovided for site-specific recombination of introduced nucleic acidsequences in host plant cell plastids. The methods of the presentinvention provide a novel means for obtaining transplastomic plants.

As used herein, transplastomic refers to a plant cell having anintroduced nucleic acid, where the introduced nucleic acid is introducedinto the plant cell plastid. The introduced nucleic acid may beintegrated into the plastid genome or may be contained in anautonomously replicating plasmid. Preferably, the nucleic acid isintegrated into the genome of the plant cell plastid.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell means “transfection” or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

In one embodiment of the present invention, constructs are provided thatallow for the site-specific recombination of a nucleic acid sequence ina host plant cell plastid. Such constructs are referred to herein asplastid expression constructs. In general, the constructs comprise atleast one DNA sequence and at least two recombining sites.

As used herein, “recombining sites” (also referred to herein as“recombination sites”) refer to nucleic acid sequences comprisinginverted palindromes separated by an asymmetric sequence at which asite-specific recombination can occur. Such recombining sites caninclude, but are not limited to, Lox (Sternberg et al. (1978) ColdSpring Harbor Symp. Quant. Biol. 43:1143-1146 and Hoess et al. (1990) InNucleic Acids and Molecular Biology, Eds Eckstein and Lilley (Springer,Berlin), vol 4, pp 99-109) and FRT (reviewed in Kilby et al. (1993)Trends In Genetics, 9, 413-421).

Any site-specific recombination system can be used in accordance withthe present invention in the plastid expression constructs. Morepreferably, any similar recombination system in which the recognitionsite for the recombinase consists of binding sites flanking anasymmetric spacer sequence can be used. Particularly preferredsite-specific recombination systems would include, but are not limitedto, the Cre/lox and FLP/FRT site-specific recombination systems. Boththe Cre recombinase derived from bacteriophage P1 and the FLPrecombinase derived from Saccharomyces cerevisiae mediate site-specificrecombination between a pair of target sequences and are members of theintegrase family. The chemical structures of over 100 members of the lntfamily of site-specific recombinases have been compared (Nunes-Duby etal., Nucl. Acids Res. 26, 2:391-406). The recombination mechanism ofthese systems has also been investigated (Craig, Annu. Rev. Genet.22:77-105, 1988; Grindley, Curr. Biol., 7:608-612, 1997).

The loxP recombination site is the target sequence for the Crerecombinase. The loxP target sequence comprises two 13-base pair (bp)inverted repeats and a central or core 8-bp sequence referred to as the“spacer region.” The Cre recombinase catalyzes a reversible reactionwherein fragments of DNA in between the wild-type loxP sites can beexcised, integrated, or exchanged in a crossover event with another DNAmolecule containing a pair of compatible lox sites. A compatible site asused herein means a recombining site that is capable of recombinationwith another recombining site. Accordingly, two wild-type loxP sites arecompatible (capable of recombination with each other). Compatible loxsites can also excise DNA as well as integrate DNA. In this regard,compatible lox sites can be useful for cloning methods in which excisionof DNA fragments is desired.

Of interest in the plastid constructs of the present invention is theuse of at least two recombining sites. In particular, where tworecombining sites are employed, the two sites are preferably compatible.Where more than two recombining sites are employed, for example fourrecombining sites, the sites will be provided in pairs. The individualrecombining sequences of each pair are preferably compatible; however,the sequences of one pair can be compatible with members of a secondpair or can be non-compatible (not capable of recombination with eachother).

The DNA sequence for use in the plastid constructs of the presentinvention can be any DNA sequence. DNA sequences of interest for use inthe plastid constructs of the present invention include, but are notlimited to, blocking sequences and expression constructs.

Blocking sequences refer to nucleic acid sequences that are locatedbetween two sequences of interest. Excision of the blocking sequencesresults in the two sequences being brought into operable association.For example, where the DNA sequence is located between a plastidfunctional promoter and a nucleic acid sequence to be expressed from thepromoter, excision of the blocking sequence results in the promoter andthe nucleic acid sequence of interest being brought together to form afunctional expression cassette. Such blocking sequences have beendescribed, for example, for use in the nuclear genome in U.S. Pat. No.5,925,808.

Also of interest for use in the plastid constructs of the presentinvention is a DNA sequence positioned between two recombining sites. Ofparticular interest is a DNA sequence comprising a plastid expressioncassette. The cassettes preferably have a plastid promoter and a DNAsequence of interest. The recombining sites can be positioned within theexpression cassette, outside the expression cassette, or combinationsthereof. Of interest in the plastid expression cassettes are nucleicacid sequences encoding genes of interest. Of particular interest is theuse of sequences encoding genes that confer resistance to herbicides orantibiotics. Of most particular interest is the use of selectablemarkers in the plastid expression cassettes.

Where the recombining sites are positioned outside the cassette, thesites can be positioned 5′ to the promoter. Positioned 5′ to thepromoter refers to a recombining site located upstream of the promoterwith respect to the direction of transcription. In addition, therecombining sites can be positioned 3′ of the DNA sequence of interest.Positioned 3′ of the DNA sequence of interest refers to the location ofthe recombining site downstream of the sequence with respect to thedirection of transcription. Such positioning 5′ or 3′ allows for theplacement of additional sequences, such as transcriptional enhancers,transcriptional termination sequences, and the like between therecombining site and the promoter and/or DNA sequence. Furthermore,recombining sites can be positioned as to place the DNA sequence betweenthe recombining sites.

Thus, where a DNA sequence is positioned between two compatiblerecombining sites, the sequence can be removed by providing a compatiblerecombinase. A compatible recombinase refers to a recombinase thatrecognizes a specific recombining sequence.

Also provided in the present invention are recombinase constructs. Suchconstructs comprise a promoter functional in a plant cell, a plastidtargeting sequence, and a nucleic acid sequence encoding a proteininvolved in recombination between particular recombining sites. Suchproteins are referred to as recombinases.

Nucleic acid sequences encoding recombinases for use in the presentinvention include any recombinase involved in recombination betweenparticular recombining sites. Preferably, the recombinase used in theconstructs of the present invention is functional towards therecombining sites employed in the plastid expression constructs. Mostpreferred in the present invention are the Cre, FLP and R recombinases.Most especially preferred is the Cre recombinase.

The Cre, FLP and R recombinases belong to the lambda integrase family ofDNA recombinases (reviewed in Kilby et al. (1993) Trends in Genetics,9:413-421; Landy (1993) Current Opinion in Genetics and Development,3:699-707; Argos et al. (1986) EMBO J., 5:433-440). The Cre and FLPrecombinases show similarities, both in terms of the types of reactionsthey carry out and in the structure of their target sites and mechanismof recombination (see, e.g., Jayaram (1994) Trends in BiologicalSciences, 19:78-82; Lee et al. (1995) J. Biolog. Chem., 270:4042-4052;Whang et al. (1994) Molec. Cell. Biolog., 14:7492-7498; Lee et al.(1994) EMBO J., 13:5346-5354; Abremski et al. (1986) J. Mol. Biol.,192:17-26; Adams et al. (1992) J. Mol. Biol., 226:661-673). Forinstance, the recombination event is independent of replication andexogenous energy sources such as ATP and functions on both supercoiledand linear DNA templates.

The Cre and FLP recombinases exert their effects by promotingrecombination between two of their target recombination sites, Lox andFrt, respectively. Both target sites are comprised of invertedpalindromes separated by an asymmetric sequence (see, e.g., Mack et al.(1992) Nucleic Acids Research, 20:4451-4455; Hoess et al. (1986) NucleicAcids Research, 14:2287-2300; Kilby et al.(l 993) supra). The asymmetryprovides directionality to the recombination event. Namely,recombination between target sites arranged in parallel (also referredto as “direct repeats”) on the same linear DNA molecule results inexcision of the intervening DNA sequence (the DNA sequence that isflanked by the recombining sites) as a circular molecule (Kilby et al.(1993) supra). Recombination between direct repeats on a circular DNAmolecule excises the intervening DNA and generates two circularmolecules. In comparison, recombination between antiparallel sites(sites that are in opposite orientation, also referred to as “invertedrepeats”) on a linear or circular DNA molecule results in inversion ofthe internal sequence. Even though recombinase action can result inreciprocal exchange of regions distal to the target site when targetsare present on separate linear molecules, intramolecular recombinationis favored over intermolecular recombination.

Any nucleic acid can be introduced into a host cell plastid by themethods encompassed by the present invention including, for example, DNAsequences or genes from another species, and/or genes or sequences thatoriginate with or are present in the same species but are incorporatedinto recipient cells by genetic engineering methods rather thanclassical reproduction or breeding techniques. An introduced piece ofDNA can be referred to as exogenous DNA. Exogenous as used herein isintended to refer to any gene or DNA segment that is introduced into arecipient cell, regardless of whether a similar gene may already bepresent in such a cell. The type of DNA included in the exogenous DNAcan include DNA that is already present in the plant cell, DNA fromanother plant, DNA from a different organism, or a DNA generatedexternally, such as a DNA sequence containing an antisense message of agene, or a DNA sequence encoding a synthetic or modified version of agene.

The constructs of the present invention find use in methods forsite-specific recombination in host plant cell plastids.

The methods of the present invention involve introducing plastidconstructs described herein into a plant cell plastid and providing thecell with a recombinase. The recombinase can be provided to thetransplastomic plant cell by a variety of methods including, but notlimited to, expressing the recombinase in a plant cell nucleus andtargeting the expressed gene to the plastid, expressing the recombinasefrom the plant cell plastid, various culture conditions, sprays, and thelike.

Of particular interest in the methods of the present invention is theuse of nucleotide sequences encoding recombinases in recombinant DNAconstructs to direct expression of the recombinase protein to a hostcell plastid. Of particular interest is the use of the polynucleotidesequences encoding recombinase in recombinant DNA constructs employing aplastid targeting sequence to direct the expressed recombinase to thehost plant cell plastid.

The recombinase expression constructs generally comprise a promoter(also referred to as a transcriptional initiation region) functional ina plant host cell operably linked to a nucleic acid sequence encoding arecombinase having a plastid targeting sequence and a transcriptionaltermination region functional in a host plant cell.

Those skilled in the art will recognize that there are a number ofpromoters that are functional in plant cells and have been described inthe literature. Chloroplast and plastid specific promoters, chloroplastor plastid functional promoters, and chloroplast or plastid operablepromoters are also envisioned.

Any plant promoter can be used as a 5′ regulatory sequence formodulation of expression of a particular gene or genes. The promoterregion contains a sequence of bases that signals RNA polymerase toassociate with the DNA and to initiate the transcription into mRNA usingone of the DNA strands as a template to make a correspondingcomplementary strand of RNA. Those of skill in the art are aware of thenumerous types of promoters that can be used in a plant expressionvector and a number of promoters that are active in plant cells havebeen described in the literature. A number of promoters have utility forplant gene expression for any gene of interest including, but notlimited to, selectable markers, scorable markers, genes for pesttolerance, disease tolerance, nutritional enhancements and any othergene of agronomic interest. Examples of constitutive promoters usefulfor plant gene expression include, but are not limited to, thecauliflower mosaic virus (CaMV) 35S promoter, which confersconstitutive, high-level expression in most plant tissues (see, e.g.,Odel et al., Nature 313:810, 1985), including monocots (see, e.g.,Dekeyser et al., Plant Cell 2:591, 1990; Terada and Shimamoto, Mol. Gen.Genet. 220:389, 1990); the nopaline synthase promoter (An et al., PlantPhysiol. 88:547, 1988) and the octopine synthase promoter (Fromm et al.,Plant Cell 1:977, 1989); and the figwort mosaic virus (FMV) promoter.Other types of promoters are also envisioned to have utility in thepresent invention including, but not limited to, tissue-enhancedpromoters, developmentally regulated promoters, or inducible promoters.

Of interest in the present invention are promoters that are functionalduring zygote formation (before seeds germinate) or subsequent seedlinggermination. Examples of such promoters include, but are not limited to,Act2 and Act8 (An et al. (1996) Plant J., 10: 107-121) and Per1(Haslekas (1998) Plant Mol Biol 36(6):833-45).

Preferably, the proteins conferring various recombinases are directed toa particular subcellular compartment, for example, to the mitochondrion,endoplasmic reticulum, vacuoles, chloroplast or other plastidiccompartment. For example, where the recombinase will be targeted toplastids, such as chloroplasts, for expression, the constructs will alsoemploy the use of sequences to direct the gene to the plastid. Suchsequences are referred to herein as chloroplast transit peptides (CTP)or plastid transit peptides (PTP). In this manner, where the gene ofinterest is not directly inserted into the plastid, the expressionconstruct will additionally contain a gene encoding a transit peptide todirect the gene of interest to the plastid. Such transit peptides areknown in the art. See, for example, Von Heijne et al. (1991) Plant Mol.Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem.264:17544-17550; della-Cioppa et al. (1987) Plant Physiol. 84:965-968;Romer et al. (1993) Biochem. Biophys. Res Commun. 196:1414-1421; and,Shah et al. (1986) Science 233:478-481.

Regulatory transcript termination regions may be provided in plantexpression constructs of this invention as well. Transcript terminationregions may be provided by any convenient transcription terminationregion derived from a gene source, for example, the transcripttermination region that is naturally associated with the transcriptinitiation region. The skilled artisan will recognize that anyconvenient transcript termination region that is capable of terminatingtranscription in a plant cell may be employed in the constructs of thepresent invention.

Alternatively, constructs may be prepared to direct the expression ofthe recombinase sequences directly from the host plant cell plastid.Such constructs and methods are known in the art and are generallydescribed, for example, in Svab et al. (1990) Proc. Natl. Acad. Sci. USA87:8526-8530 and Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA90:913-917 and in U.S. Pat. No. 5,693,507.

A plant cell, tissue, organ, or plant into which the recombinant DNAconstructs containing the expression constructs have been introduced isconsidered transformed, transfected, or transgenic. A transgenic ortransformed cell or plant also includes progeny of the cell or plant andprogeny produced from a breeding program employing such a transgenicplant as a parent in a cross and exhibiting an altered phenotyperesulting from the presence of a nucleic acid sequence.

The constructs of the present invention can be employed with a widevariety of plant life. Plants of interest include, but are not limitedto, rapeseed (Canola and High Erucic Acid varieties), sunflower,safflower, Arabidopsis, rice, cotton, soybean, peanut, coconut and oilpalms, wheat, and corn. Depending on the method for introducing therecombinant constructs into the host cell, other DNA sequences may berequired. Importantly, this invention is applicable to dicotyledonousand monocotyledonous species alike and will be readily applicable to newand/or improved transformation and regulation techniques.

The method of transformation in obtaining such transgenic plants is notcritical to the instant invention, and various methods of planttransformation are currently available. Furthermore, as newer methodsbecome available to transform crops, they may also be directly appliedhereunder. For example, many plant species naturally susceptible toAgrobacterium infection may be successfully transformed via tripartiteor binary vector methods of Agrobacterium-mediated transformation. Inmany instances, it will be desirable to have the construct bordered onone or both sides by T-DNA, particularly having the left and rightborders, more particularly the right border. This is particularly usefulwhen the construct uses A. tumefaciens or A. rhizogenes as a mode fortransformation, although the T-DNA borders may find use with other modesof transformation. In addition, techniques of microinjection, DNAparticle bombardment, and electroporation have been developed that allowfor the transformation of various monocot and dicot plant species.

Normally, included with the DNA construct will be a structural genehaving the necessary regulatory regions for expression in a host andproviding for selection of transformant cells. The gene may provide forresistance to a cytotoxic agent, e.g., antibiotic, heavy metal, toxin,etc.; complementation providing prototrophy to an auxotrophic host;viral immunity or the like. Depending upon the number of different hostspecies the expression construct or components thereof are introducedinto, one or more markers may be employed, where different conditionsfor selection are used for the different hosts.

Where Agrobacterium is used for plant cell transformation, a vector maybe used that may be introduced into the Agrobacterium host forhomologous recombination with T-DNA or the Ti- or Ri-plasmid present inthe Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNA forrecombination may be armed (capable of causing gall formation) ordisarmed (incapable of causing gall formation), the latter beingpermissible, so long as the vir genes are present in the transformedAgrobacterium host. The armed plasmid can give a mixture of normal plantcells and gall.

In some instances where Agrobacterium is used as the vehicle fortransforming host plant cells, the expression or transcription constructbordered by the T-DNA border region(s) will be inserted into a broadhost range vector capable of replication in E. coli and Agrobacterium,there being broad host range vectors described in the literature.Commonly used is pRK2 or derivatives thereof. See, for example, Ditta etal. (Proc. Nat. Acad. Sci., U.S.A. (1980) 77:7347-7351) and EPA 0 120515, which are incorporated herein by reference. Alternatively, one mayinsert the sequences to be expressed in plant cells into a vectorcontaining separate replication sequences, one of which stabilizes thevector in E. coli. and the other in Agrobacterium. See, for example,McBride and Summerfelt (Plant Mol. Biol. (1990) 14:269-276), wherein thepRiHRI (Jouanin et al., Mol. Gen. Genet. (1985) 201:370-374) origin ofreplication is utilized and provides for added stability of the plantexpression vectors in host Agrobacterium cells.

Included with the expression construct and the T-DNA will be one or moremarkers, which allow for selection of transformed Agrobacterium andtransformed plant cells. A number of markers have been developed for usewith plant cells, such as resistance to chloramphenicol, kanamycin, theaminoglycoside G418, hygromycin, or the like. The particular markeremployed is not essential to this invention, one or another marker beingpreferred depending on the particular host and the manner ofconstruction.

For transformation of plant cells using Agrobacterium, explants may becombined and incubated with the transformed Agrobacterium for sufficienttime for transformation, the bacteria killed, and the plant cellscultured in an appropriate selective medium. Once callus forms, shootformation can be encouraged by employing the appropriate plant hormonesin accordance with known methods and the shoots transferred to rootingmedium for regeneration of plants. The plants may then be grown to seedand the seed used to establish repetitive generations and for isolationof vegetable oils.

In order to provide a means of selecting the desired plant cellsfollowing plastid transformation, the polynucleotides for plastidtransformation will also contain a construct that provides forexpression of a marker gene. Expression of the marker gene productallows for selection of plant cells comprising plastid organelles thatare expressing the marker protein. In the examples provided herein, abacterial aadA gene is expressed under the regulatory control ofchloroplast 5′ promoter and 3′ transcription termination regions. Theuse of such an expression construct for plastid transformation of plantcells has been described by Svab and Maliga (1993, supra). Expression ofthe aadA gene confers resistance to spectinomycin and streptomycin andthus allows for the identification of plant cells expressing this markergene. Selection for the aadA marker gene is based on identification ofplant cells that are not bleached by the presence of streptomycin, ormore preferably spectinomycin, in the plant growth medium. Other genesthat encode a product involved in chloroplast metabolism may also beused as selectable markers. For example, genes that provide resistanceto plant herbicides such as glyphosate, bromoxynil or imidazolinone mayfind particular use. Such genes have been reported by Stalker et al. (J.Biol. Chem. (1985) 260:4724-4728; glyphosate resistant EPSP), Stalker etal. (J. Biol. Chem. (1985) 263:6310-6314; bromoxynil resistant nitrilasegene), and Sathasivan et al. (Nucl. Acids Res. (1990) 18:2188; AHASimidazolinone resistance gene).

In the examples described herein, the aadA gene is under the control ofa tobacco psbA gene promoter, PpsbA. Numerous additional promoterregions may also be used to drive expression of the selectable markergene, including various plastid promoters, viral and bacterial promotersthat have been shown to function in plant plastids.

The polynucleotides for use in plastid transformation will also containa means of providing for stable transfer of the expression construct andthe selectable marker construct into the plastid genome. Conveniently,regions of homology to the target plastid genome flank the constructs tobe transferred and provide for transfer to the plastid genome byhomologous recombination via a double crossover into the genome. Wherethe regions of homology are present in the inverted repeat regions (IRAand IRB) of the plastid genome, two copies of the transgene are expectedper plastid genome. Typically, the regions of homology with the plastidgenome will be approximately 1 kb in size. Smaller regions of homologymay also be used, for example as little as 100 bp can provide forhomologous recombination into the plastid genome. However, the frequencyof recombination and thus the frequency of obtaining plants havingtransformed plastids may decrease with decreasing size of the homologyregions. Example of constructs comprising such regions of homology fortobacco plastid transformation are described in Svab et al. (1990 supra)and Svab and Maliga (1993 supra). Regions useful for recombination intotobacco plastid genomes are also described in the following examples.Similar homologous recombination and selection constructs may beprepared using plastid DNA from the target plant species.

In developing the constructs of the instant invention, the variousfragments comprising the regulatory regions and open reading frame maybe subjected to different processing conditions, such as ligation,restriction enzyme digestion, PCR, in vitro mutagenesis, linkers andadapters addition, and the like. Thus, nucleotide transitions,transversions, insertions, deletions, or the like, may be performed onthe DNA that is employed in the regulatory regions, the recombinaseencoding sequence and/or the DNA sequences of interest for expression inthe plastids. Methods for restriction digests, Klenow blunt endtreatments, ligations, and the like are well known to those in the artand are described, for example, by Maniatis et al. (in Molecularcloning: a laboratory manual (1982) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.).

During the preparation of the constructs, the various fragments of DNAwill often be cloned in an appropriate cloning vector, which allows foramplification of the DNA, modification of the DNA or manipulation byjoining or removing of sequences, linkers, or the like. Normally, thevectors will be capable of replication in at least a relatively highcopy number in E. coli. A number of vectors are readily available forcloning, including such vectors as pBR322, pUC series, M13 series, andpBluescript (Strategene; La Jolla, Calif.).

Stable transformation of tobacco plastid genomes by particle bombardmenthas been reported (Svab et al. (1990) Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA90:913-917). The methods described in the above references may beemployed to obtain plants transformed with the plastid transformationconstructs described herein. Briefly, such methods involve DNAbombardment of a target host explant, preferably from a tissue that isrich in metabolically active plastid organelles, such as green planttissues, including leaves, and cotyledons. The bombarded tissue is thencultured for ˜2 days on a cell division promoting media. The planttissue is then transferred to a selective media containing an inhibitoryamount of the particular selective agent, as well as the particularhormones and other substances necessary to obtain regeneration for thatparticular plant species. For example, in the above publications and theexamples provided herein, the selective marker is the bacterial aadAgene and the selective agent is spectinomycin. The aadA gene productallows for continued growth and greening of cells whose chloroplastscomprise the marker gene product. Cells that do not contain the markergene product are bleached. The bombarded explants will form green shootsin approximately 3-8 weeks. Leaves from these shoots are thensubcultured on the same selective media to ensure production andselection of homoplasmic shoots. As an alternative to a second round ofshoot formation, the initial selected shoots may be grown to matureplants and segregation relied upon to provide transformed plantshomoplastic for the inserted gene construct.

The transformed plants so selected may then be analyzed to determinewhether the entire plastid content of the plant has been transformed(homoplastic transformants). Typically, following two rounds of shootformation and spectinomycin selection, approximately 50% of thetransgenic plantlets analyzed are homoplastic as determined by Southernblot analysis of plastid DNA. These plantlets are selected for furthercultivation, both for analysis of the transgenic plastid phenotype, orfor use in methods to re-transform the transplastomic plants with therecombinase construct.

The constructs of the present invention provide a novel means for theproduction of plants having transformed plastids. The constructs providemethods for the introduction of polynucleotides into a plant cellplastid. In this manner, transplastomic plants can be obtained in whicha particular plastid expression cassette has been removed by the actionof the expressed recombinase on the recombining sites. For example, whenthe plastid expression cassette flanked by the recombining sitescontains a selectable marker, the resulting plants can then be used insubsequent rounds of plastid transformation using the same marker forselection of transplastomic plants.

Thus, the present invention provides methods for the production oftransplastomic plants. The methods involve the production of plantshaving an introduced polynucleotide that is removed when contacted withan appropriate recombinase. In general, the polynucleotides for use inthe methods will be flanked by recombining sites that are in a parallelorientation.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedfor purposes of illustration only and are not intended to limit thepresent invention.

EXAMPLES Example 1 Expression Construct Preparation

Expression constructs are prepared to allow the excision of anintroduced marker gene from the plastid genome when expressed in thesame plant expressing a recombinase directed to the plastid. The markergene is flanked by the loxP sequences (5′-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3′ (SEQ ID NO:1), the spacer region is providedas an underline). In addition constructs are prepared to direct theexpression of a recombinase from the nuclear genome, and direct theprotein to the plastid.

1A. Plastid Expression Constructs

A series of plastid expression constructs are prepared for use inplastid transformations. The constructs are prepared using the aadAselectable marker flanked by directly repeated loxP sites. A schematicdiagram of the aadA selectable marker cassette is provided in FIG. 1.FIG. 1 also provides the sequence of the loxP recombining sites. Theconstructs also employ the use of the Green Fluorescent Protein (GFP)expressed from promoter sequences functional in a plant cell plastid.

The nucleic acid sequence encoding GFP-2 as described by Pang et al.((1996) Plant Physiol., 112:893-900) was cloned between the Prrnpromoter/rbcL ribosome binding site and Trps16 transcription terminationsequence. The Prrn promoter with the synthetic ribosome binding site andtranslationally fused to sequence encoding 7 amino acids of the rbcLcoding region is as described in Svab et al. (1993, supra). The Trps16fragment comprises the rps16 gene 3′-regulatory region from nucleotides5,087 to 4,939 in the tobacco plastid DNA.

A plastid expression construct, pMON53117 (FIG. 2), was preparedemploying the aadA gene under the regulatory control of the psbApromoter and transcriptional terminator. This aadA cassette is flankedby direct repeats of the loxP recombining sites. The expressionconstruct also contains a second cassette containing the GFP sequenceunder the regulatory control of the rrn promoter and rps16transcriptional termination region. These two expression 3 0 cassettesare inserted into the transformation construct pPRV111B (Zoubenko et al.(1994) Nucleic Acids Research, 22(19):3819-3824). FIG. 2 provides aschematic diagram of the pMON53117 construct.

A second plastid expression construct, pMON53119 (FIG. 3), was alsoprepared. This construct is generally the same as described forpMON53117; however, the aadA cassette is cloned between the rrn promoterand the coding sequence of GFP such that expression of the GFP markergene requires the excision of the aadA cassette by the Cre recombinase.The direction of the aadA cassette is counter to the direction of GFPexpression to further ensure that excision of the aadA cassette isnecessary for GFP expression. The construct is designed such thatexcision of the aadA cassette creates a translationally fused lox sitein frame with the GFP protein. A schematic diagram of the pMON53119construct is provided in FIG. 3.

Prior to use in plastid transformation, these constructs were tested inan E. coli strain that constitutively expressed the Cre recombinase.Plasmid DNA can be rescued from the transformed E. coli and restrictiondigest analysis can be used to confirm that excision occurred accuratelyand completely. This analysis confirmed that excision occurred properlyfor both plastid expression constructs.

1B. Recombinase Constructs

A series of constructs were prepared to direct the expression of the Crerecombinase from the plant cell nucleus and targeted to the plant cellplastid. These constructs employ the use of a plastid transit peptide todirect the recombinase to the plant cell plastid.

The construct pMON49602 (FIG. 4) is a double border plant transformationvector containing the Cauliflower Mosaic Virus 35S promoter (CaMV 35S),a chloroplast transit peptide (CTP) from the Arabidopsis thaliana EPSPsynthase gene (Klee et al., 1987, Mol Gen Genet 210: 437-442), a nucleicacid sequence encoding the bacteriophage P1 Cre recombinase, nopalinesynthase transcriptional termination sequence, as well as the kanamycinresistance gene under the regulatory control of the nopaline synthasepromoter and transcriptional termination region.

The construct pMON53147 (FIG. 5) is a double border plant transformationconstruct containing the Arabidopsis Actin2 promoter and intron (An etal. (1996) Plant J 10(1):107-21) controlling the expression of theCTP-Cre recombinase fusion, and nopaline synthase transcriptionaltermination sequence, as well as the kanamycin resistance gene under theregulatory control of the CaMV 35S promoter and nopaline synthasetranscriptional termination region.

The construct pMON49608 (FIG. 6) is a double border plant transformationconstruct containing the Arabidopsis Per1 promoter (Haslekas et al.(1998) Plant Mol Biol 36(6):833-45) for expression of the CTP-Crerecombinase fusion, and the nopaline synthase transcriptionaltermination region, as well as the kanamycin resistance gene under theregulatory control of the CaMV 35S promoter and nopaline synthasetranscriptional termination region.

The Cre recombinase sequence employed in all of the above constructscontains an intron sequence from the potato ST-LS 1 gene (Vancanneyt etal., 1990, Mol Gen Genet 220:245-250) to prevent possible complicationsas a result of expression of the Cre protein in bacterial cells duringcloning and manipulations.

Example 2 Plant Transformation

2A. Nuclear Transformation

Tobacco plants transformed to express the constructs pMON49602,pMON53147 and pMON49608 in the nucleus of a plant cell may be obtainedas described by Horsch et al. (Science (1985) 227:1229-1232).

2B. Plastid Transformation

Tobacco plastids are transformed by particle gun delivery ofmicroprojectiles as described by Svab and Maliga (Proc. Natl. Acad. Sci.(1993) 90:913-917), and described herein.

Dark green, round leaves are cut, preferably from the middle of theshoots, from 3-6-week-old Nicotiana tabacum cv. Havana that have beenmaintained in vitro on hormone-free MS medium (Murashige and Skoog(1962) Physiol Plant. 15, 473-497) supplemented with B5 vitamins inPhytatrays or sundae cups with a 16-hour photoperiod at 24° C. Each cutleaf is then placed adaxial side up on sterile filter paper over tobaccoshoot regeneration medium (TS0 medium: MS salts, 1 mg/LN⁶-benzyladenine, 0.1 mg/L 1-naphthaleneacetic acid, 1 mg/L thiamine,100 mg/L inositol, 7 g/L agar pH 5.8 and 30 g/L sucrose). Leaves arepreferably placed in the center of the plate with as much contact withthe medium as possible. The plates are preferably prepared immediatelyprior to use, but they may be prepared up to a day before transformationby particle bombardment by wrapping in plastic bags and storing at 24°C. overnight.

Tungsten or gold particles are sterilized for use as microcarriers inbombardment experiments. Particles (50 mg) are sterilized with 1 mL of100% ethanol and stored at −20° C. or −80° C. Immediately prior to use,particles are sedimented by centrifugation, washed with 2 to 3 washes of1 mL sterile deionized distilled water, vortexed and centrifuged betweeneach wash. Washed particles are resuspended in 500 μL 50% glycerol.

Sterilized particles are coated with DNA for transformation. Twenty-fivemicroliter aliquots of sterilized particles are added to a 1.5-mLmicrofuge tube, and 5 μg of DNA of interest is added and mixed bytapping. Thirty-five microliters of a freshly prepared solution of 1.8MCaCl₂ and 30 mM spermidine is added to the particle/DNA mixture, mixedgently, and incubated at room temperature for 20 minutes. The coatedparticles are sedimented by centrifuging briefly. The particles arewashed twice by adding 200 μL 70% ethanol, mixing gently, andcentrifuging briefly. The coated particles are resuspended in 50 μL of100% ethanol and mixed gently. Five to ten microliters of coatedparticles are used for each bombardment.

Transformation by particle bombardment is carried out using the PDS 1000Helium gun (Bio Rad, Richmond, Calif.) using a modified protocoldescribed by the manufacturer.

Plates containing the leaf samples are placed on the second shelf fromthe bottom of the vacuum chamber and bombarded using the 1100 p.s.i.rupture disk. After bombardment, petri plates containing the leafsamples are wrapped in plastic bags and incubated at 24° C. for 48hours.

After incubation, bombarded leaves are cut into approximately 0.5 cm²pieces and placed abaxial side up on TSO medium supplemented with 500μg/mL spectinomycin. After 3 to 4 weeks on the selection medium, small,green spectinomycin resistant shoots will appear on the leaf tissue.These shoots will continue to grow on spectinomycin containing mediumand are referred to as primary putative transformants.

When the primary putative transformants have developed two to threeleaves, two small pieces (approximately 0.5 cm²) are cut from each leafand used for either selection or for a second round of shootregeneration. One piece is placed abaxial side up on plates containingTSO medium supplemented with 500 μg/mL spectinomycin, and the otherpiece is placed abaxial side up on TSO medium supplemented with 500μg/mL each of spectinomycin and streptomycin. Positive transformants areidentified as the shoots that form green callus on the TSO mediumcontaining spectinomycin and streptomycin.

After 3 to 4 weeks, the tissue placed on TSO medium containing onlyspectinomycin, which has been identified as positive on the TSO mediumwith spectinomycin and streptomycin, will develop green shoots. Two tofour shoots of each positive transformant are selected and transferredto TSO medium supplemented with 500 μg/mL spectinomycin for generationof roots. Southern analysis is performed on two shoots to confirmhomoplasmy as described below. Shoots from homoplasmic events aretransferred to the greenhouse for seed production, whereas transformantsthat are not homoplasmic are sent through a second round of regenerationon TSO medium with 500 μg/mL spectinomycin to attain homoplasmy.

Example 3 In Vivo Excision of the aadA Marker Gene

For the excision of the aadA marker gene expression cassette flanked bythe loxP sites, a variety of methods can be employed. One methodinvolves the use of homoplasmic pMON53117 and pMON53119 transformants inretransformation experiments with the nuclear recombinase constructs.

For example, homoplasmic plastid transformants derived from plastidvector pMON53119 (Nt-53119 lines) carrying the disrupted GFP gene can beused as recipient for re-transformation. The nuclear re-transformationvector pMON49602 carrying the constitutive 35S::CTP-Cre recombinase genecan be introduced via Agrobacterium-mediated nuclear transformation andselection for kanamycin resistance encoded in the transformation vector.The CTP provides for the localization of the Cre gene product into theplastids for excision of the aadA gene cassette. Transformed lines thathave excised the plastid aadA gene can be identified by kanamycinresistance and GFP fluorescence and spectinomycin and/or streptomycinsusceptibility.

In this example, excision of aadA can occur from all of the recipient'sNt-53119 plastid genomes or only a fraction of these. Therefore, GFPfluorescence can be observed either completely throughout the newlyregenerating plantlet or only in sectors of the regenerant. Shoots thatshow complete or nearly complete GFP fluorescence can be analyzedfurther and grown to maturity.

The nuclear CTP-Cre and selectable marker genes can also be removed fromthe re-transformed Nt-53119 plants by genetic segregation in T1seedlings. Pollen from wild-type plants can be used to pollinate thenuclear re-transformed Nt-53119 plants. Because the nuclearre-transformed lines are hemizygous, the seedlings generated from theback-cross segregate the nuclear transgenes in a 2 (wild-type):2(transgene+) ratio. Because the re-transformed Nt-53119 plant is used asmaternal parent, all of the progeny will have the plastid transgene ofinterest. Therefore, the final result is ½ of the progeny that carry theplastid GFP gene without the plastid aadA selectable marker and also donot carry any nuclear transgenes.

Loss of the nuclear transgenes can be monitored by germinating seeds onmedium containing kanamycin monosulfate (100 mg/L). The seedlings thatcompletely bleach on kanamycin have segregated out the nuclear nptIIselectable marker and the CTP-Cre genes. Bleached seedlings can berescued onto drug-free medium and screened for spectinomycin sensitivityand GFP fluorescence. Southern analysis can be performed to confirm thefinal GFP+, aadA−, nptII− transgenic plants.

Another method for the in vivo excision of the aadA expression cassetteinvolves the crossing of plastid transformed lines to nucleartransformed CTP-Cre recombinase lines.

Nuclear transformants carrying CTP-Cre recombinase genes can begenerated via Agrobacterium-mediated transformation using kanamycinselection encoded on the nuclear transformation vector. Independentlines for each of the different CTP-Cre constructs (35S::CTP-Cre,Act2::CTP-Cre, Per1::CTP-Cre) can be generated. These lines arecharacterized to identify single copy inserts to be used in subsequentcrosses to the plastid transformed lines. Each of these nucleartransformed lines is hemizygous for the nuclear transgenes.

For example, homoplasmic plastid transformants, obtained from vectorspMON53117 and pMON53119, can be cross pollinated with the hemizygousnuclear CTP-Cre lines. Alternatively, transplastomic lines containingpMON53117 and pMON53119 can be used in transformation to obtain lineshaving both a plastid construct and a recombinase construct. The nucleartransformed lines are used as pollen donor; the plastid transformedlines are used as maternal recipient. CTP-Cre gene product is importedinto plastids for excision of the aadA gene. This excision can occurduring zygote formation (before seeds germinate) or subsequent seedlinggermination, depending on the activity of the promoter used to drive thenuclear CTP-Cre gene. The Act2 and Per1 promoters are reported to beactive during fertilization, therefore excision occurs early indevelopment. The 35S promoter is constitutive and active during seedlingdevelopment.

Early expression of the CTP-Cre and excision during zygote formation ofthe sequence flanked by the loxP allows deletion of the aadA gene in allor most of the plastid genome copies.

Furthermore, because the nuclear transformed lines are hemizygous, a 2(wild-type) to 2 (nuclear transgene+) segregation can occur in theseedlings. Seeds can be germinated on medium containing kanamycinmonosulfate (100 mg/L) to identify wild-type nuclear background. Tissuesthat bleach on kanamycin will then be screened for spectinomycinsensitivity (aada excision) and GFP expression. Transformants derivedfrom pMON53117 will be uniformly GFP positive, whereas transformantsderived from pMON53119 will only be GFP positive if the aadA gene isexcised from at least one chloroplast genome. Southern analysis will beperformed to confirm transgenic plants.

If excision occurs later in the development of seedlings, the deletionof the aadA gene will occur in only a partial population of the plastidgenome copies. These will be identified as F1 plants that express bothspectinomycin resistance and GFP. In this case, kanamycin-sensitive,GFP+ lines will be grown to maturity again and allowed to set self seed.Homoplasmic plants that no longer carry any aadA gene copies can beidentified in the subsequent next generation.

Example 4 aadA Disrupting GFP; Nuclear Retransformation

Line Nt-53119 was generated from plastid transformation of wild-typetobacco with plasmid pMON53119 using standard microprojectilebombardment protocols and spectinomycin selection as described inExample 2. Primary plastid transformed Nt-53119 lines were verified bySouthern blot analysis and were initially heteroplasmic. Plants wereregenerated from leaf samples of the primary transformed Nt-53119 lineuntil homoplasmic plants were identified. The homoplasmicplastid-transformed Nt-53119 lines carry the inactive GFP gene due todisruption by aadA (flanked by lox sites). Under fluorescent microscopy,these lines appear red due to chlorophyll autofluorescence. HomoplasmicNt-53119 lines were subsequently used for nuclear retransformations byAgrobacterium.

Agrobacterium nuclear retransformation of Nt-53119 plastid lines wasperformed according to standard procedures. Two different binary vectorswere used for the retransformations: Plasmid pMON49602 and pMON53147carrying the CTP-Cre gene driven by the CAMV 35S and the Arabidopsisthaliana Act2 promoters, respectively. These binary vectors also carrythe nptlI gene encoding resistance to kanamycin used as the selectablemarker for the retransformations.

Kanamycin-resistant nuclear retransformed lines were screened by visualobservation for GFP fluorescence, which indicates excision of the aadAgene from the disrupted GFP gene, thus re-activating GFP function. AfterAgrobacterium transformation of the Nt-53119 line, numerous GFP positiveshoots were observed from transformations with binary vectors carryingCTP-Cre driven by either the CAMV 35S or the Act2 promoter. These dataindicate that CTP-Cre can be properly targeted to plastids and markerexcision can occur early during shoot development.

GFP+ shoots from multiple independently retransformed lines wereisolated and moved to individual tissue culture plates for continuedgrowth. The independent nuclear retransformed shoots were assayed forthe presence of the plastid transgenes aadA and GFP by Southern blotanalysis. Leaf pieces from young shoots were dissected, and totalcellular DNA was prepared. Total cellular DNA was digested with BamHIand probed with plastid DNA flanking the transgene insertion site. Bythis analysis, the size of the insertion in the plastid genome can bedetermined and therefore the presence of the GFP gene and absence ofaadA can be molecularly visualized.

Multiple representative nuclear retransformed Nt-53119 lines derivedfrom Agrobacterium transformation with the pMON53147 binary vectorcarrying the Act2-promoter driving CTP-Cre were analyzed by Southernblots using plastid DNA flanking the insertion site as probe. Theselines are referred to as Nt-Act2-53119—followed by the clone number.Although the wild-type untransformed tobacco control has a hybridizingband at the predicted 3.27 kb size, the parental Nt-53119 plastid lineprior to nuclear retransformation has a single hybridization signal atthe size expected for the integrated aadA and GFP genes (unexcised; 5.58kb). All Nt-Act2-53119—lines (for example, lines Nt-Act2-531194, 9, 11,12, 14, 16, 20, 23, 25, 38, 40, and 43) carry a strong hybridizing bandat the size of 4.34 kb as expected for the excision of the aadA gene.When the blot is stripped and reprobed with the aadA coding region,there is no hybridization signal to nearly all of the nuclearretransformed lines. This result shows that these lines have completelylost the aadA gene from all of the plastid genomes, indicating thatCTP-Cre activity is efficient for removal of genes from the multipleplastid genomes per cell. Only one exception is seen in lineNt-Act2-53119-43, which still carries some aadA-containing plastidgenomes, as evidenced by a higher parental molecular weight band and acorresponding band when probed with aadA.

A few of the Nt-Act2-53119—lines carried an additional one or twohybridizing bands that may represent alternate aadA excision events fromthe plastid genome. One band was interemediate in size between the sizepredicted for the wild-type fragment and the correctly excised fragment,whereas the other band was smaller in size than the predicted wild-typesize. To investigate these events further, subclones of these nuclearretransformed lines were generated by a new round of plant regeneration.Multiple subclones of each line were analyzed for segregation tohomoplasmy of the putative alternate excision events. Representativesubclones were analyzed by Southern blot hybridization. The twodifferent classes of putative alternate excision events could bepurified to homoplasmy in subclones. For example, Nt-Act2-53119-38A,B,Cand Nt-Act2-53119-40C carry the intermediate-sized band in a homoplasmicform. The Nt-Act2-53119-14B,C lines carry the smaller-sized band in ahomoplasmic form. In contrast, the original Nt-Act2-53119-27 line thatcarried exclusively the correct excision event still carries only thecorrect excision event in its subclones (Nt-Act2-53119-27A,B,C). Thislatter result suggests that alternate excision events occurred earlyduring development of the nuclear retransformed lines, which are thenstable and no longer undergo alternate excision events during normalgrowth.

Multiple representative nuclear retransformed Nt-53119 lines afterAgrobacterium transformation with the pMON49602 binary vector carryingthe 35S-promoter driving CTP-Cre were also analyzed by Southern blots.Results are analogous to those observed with the pMON53147 binaryvector. The retransformed lines are referred to as Nt-35S-53119—linenumber. Nearly all of the nuclear retransformed lines analyzed (forexample, Nt-35S-53119-9, 10, 11, 12, 14, and 18) carried the correctsized 4.34 kb excised band when probed with the flanking region probe.Re-probing with the aadA coding region confirmed the absence of the aadAgene in all of these lines. In addition to the correct excision band,lines Nt-35S-53119-10, Nt-35S-53119-11 and Nt-35S-53119-18 also carriedthe alternate excision band(s). Interestingly, line Nt-35S-53119-2carried only the smaller alternate excision band and no precise 4.34 kbexcision band.

Subclones of the Nt-35S-53119 lines were generated and analyzed todetermine if the alternate excision events could be purified tohomoplasmy. The Nt-35S-53119-10A subclone carried predominantly theintermediate-sized excision event, whereas the Nt-35S-53119-10B,C andNt-35S-53119-2A,B,C subclones carried the smaller alternate excisionband in a homoplasmic form. In contrast, the Nt-35S-531 19-11 line thatcarried both correct excision and alternate excision bands now carriesexclusively the correct excision band in its subclones,Nt-35S-53119-11A,B,C. This latter result indicates that the correctexcision event can be purified to homoplasmy even if the originaltransformant carried alternate excision events.

The above results indicate that CTP-Cre is capable of efficient excisionof genes from the multiple plastid genomes per cell after nuclearretransformation into established plastid transformed lines. Excision isefficient when the CTP-Cre gene is driven by a constitutive promoter(35S) or the meristem-specific promoter (Act2). Although alternateexcision events can also occur in both cases, these are relativelyinfrequent compared to correct excision events and apparently only occurearly in development of the nuclear transformed line. Furthermore, thealternate excision events can be purified to homoplasmy if desired, insubclones of the original nuclear retransformed lines. Likewise, thecorrect excision events can be purified to homoplasmy in cases where theoriginal transformant had a mix of correct and alternate excisionevents.

Molecular Characterization of Alternate Excision Events:

PCR was used to isolate DNA fragments spanning the alternate excisionsites for both the intermediate and smaller sized bands. PCR primersthat flank the insertion site of the foreign genes in Nt-53119 lineswere used: one primer in the coding region of the 16SrRNA gene (SEQ IDNO:2) and the other primer near to the rps7/3′-rps12 coding region (SEQID NO:3). Plant lines Nt-Act2-53119-38 and Nt-35S-53119-2 were used toisolate the alternate excisions. The PCR fragments were cloned into apUC vector and DNA was subsequently prepared for sequencing thealternate excision events.

FIG. 7 shows the results of the PCR and sequencing analysis of lineNt-Act2-53119-38. The intermediate-sized excision event apparentlyoccurred through recombination of one lox site flanking the aadA geneand an alternate site (SEQ ID NO:4) in the plastid genome. The alternatesite resides downstream of the promoter of the rps7/3′-rps 12 gene, 512nucleotides from the transgene insertion site. The alternate excisionevent therefore deleted the aadA gene plus 512 bp of the residentplastid genome, resulting in a transplastomic genome carrying thetransgenic GFP gene without its promoter. Note that deletion of the 512nt of resident plastid DNA apparently has no phenotypic consequence, ashomoplasmic plants regenerated from this line show no aberrantphenotype. The sequence of the recombinant junction (SEQ ID NO:5) isshown in FIG. 7 b.

FIG. 8 shows the results of the PCR and sequencing analysis of lineNt-35S-53119-2. The smaller-sized alternate excision event apparentlyoccurred through recombination between the identical, repeated Prrnsequences; one Prrn driving expression of the GFP transgene and theother Prrn driving the expression of the endogenous 16SrRNA gene. Thedirect repeat orientation of these two Prrn sequences caused a deletionof the intervening transgenic aadA and GFP genes as well as theendogenous plastid trnV gene. The resultant plastid genome thereforedoes not carry any transgenes and is deficient for the endogenous trnVgene normally located in this region of the genome. Interestingly, thereis no apparent phenotypic consequence of trnV gene. At this time, we donot know why alternate excision through the directly repeated Prrnsequences occurred. We speculate that the close proximity of the loxsites and Cre protein stimulated homologous recombination between thedirectly repeated Prrn sequences in that local region of the plastidgenome.

Example 5 Maternal Inheritance of the Plastid GFP Transgene andMendelian Segregation of the Nuclear-Encoded Transgenes

For utility in biotechnology applications, it is desirable to remove thenuclear-encoded transgenes (nptII and CTP-Cre) from seed progeny bysegregation. To this end, primary transformed lines and subclones fromNt-Act2-53119 and Nt-35S-53119 lines were grown to maturity in thegreenhouse and allowed to self-pollinate. Because plastids arematernally inherited, the plastid-encoded GFP transgene should bepresent in all seed progeny of self pollination. On the other hand, thenuclear-encoded transgenes should segregate according to Mendelianratios and therefore be present only in a subset of the seed progeny.

To test inheritance and segregation of the transgenes, self seed wasgerminated on three different medias, all based on TSO media.Spectinomycin media (500 μg/mL) was used to detect if anyplastid-encoded aadA gene remained after excision. Kanamycin media (100μg/mL) tested for the presence of the nuclear-encoded nptII gene. Mediawithout antibiotic was used as control.

As an example of this analysis, seedlings from line Nt-Act2-53119-12 aredescribed. A Southern blot analysis was performed on randomly chosenseedlings picked from germination medium without antibiotic. All ofthese seedlings show the hybridization pattern expected from completeexcision of the aadA gene and subsequent maternal inheritance of theplastid-encoded GFP in a homoplasmic state. As expected, seeds from thisline were uniformly sensitive to spectinomycin, confirming complete lossof the aadA gene.

Seeds from the Nt-Act2-53119-12 line were also sown on medium containingkanamycin to test for the nuclear-encoded nptII gene. As expected for aMendelian inherited trait, ˜50% of the selfed seed progeny weresensitive (bleached) to kanamycin. This analysis indicates that we havesucceeded in generating plastid-transformed lines that do not carry thenuclear-encoded transgenes.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim.

1. A recombinant nucleic acid sequence comprising: a plastid constructcomprising, at least one DNA sequence, and at least two recombiningsites.
 2. The recombinant nucleic acid sequence according to claim 1,wherein said at least one DNA sequence is a first DNA sequence and asecond DNA sequence, and wherein said recombining sites are positionedbetween said first and said second DNA sequences.
 3. The recombinantnucleic acid sequence according to claim 1, wherein said at least tworecombining sites is a first recombining site and a second recombiningsite, and wherein said at least one DNA sequence is positioned betweensaid first and said second recombining sites.
 4. The recombinant nucleicacid construct according to claim 1, wherein each of said recombiningsites are selected from the group consisting of Lox, FRT, and R.
 5. Therecombinant nucleic acid construct according to claim 1, furthercomprising regions of homology for integration into the plastid genome.6. A plant cell comprising the construct according to claim
 1. 7. Aplant comprising the plant cell according to claim
 6. 8. A recombinantnucleic acid construct comprising, in the 5′ to 3′ direction oftranscription: a transcriptional initiation region functional in a plantcell, an organelle targeting sequence, and a nucleic acid sequenceencoding recombinase.
 9. The recombinant nucleic acid constructaccording to claim 8, wherein said transcriptional initiation region isselected from the group consisting of a transcriptional initiationregion functional during zygote formation, and a transcriptionalinitiation region functional during seed germination.
 10. Therecombinant nucleic acid construct according to claim 8, wherein saidtargeting sequence directs the recombinase to the plant cell plastid.11. The recombinant nucleic acid construct according to claim 8, whereinsaid recombinase is a bacteriophage P1 Cre recombinase.
 12. A method forthe production of a plant having transformed plastids, comprising:introducing into a plant cell a first recombinant DNA sequencecomprising a plastid construct comprising at least one DNA sequence, andat least two recombining sites, providing a recombinase to said plantcells, and regenerating a plant having at least one plant cellcontaining said first DNA construct.
 13. The method according to claim12, wherein said recombinase is provided to said plant cells byintroducing a second recombinant comprising: a transcriptionalinitiation region, an organelle targeting region, and a nucleic acidsequence encoding recombinase.
 14. The method according to claim 12,wherein said at least one DNA sequence is a first DNA sequence and asecond DNA sequence, and wherein said recombining sites are positionedbetween said first and said second DNA sequences.
 15. The methodaccording to claim 12, wherein said at least two recombining sites is afirst recombining site and a second recombining site, and wherein saidat least one DNA sequence is positioned between said first and saidsecond recombining sites.
 16. The method according to claim 15, whereinsaid first and said second recombining sites are parallel, i.e., indirectly repeated orientation.
 17. The method according to claim 16,whereby excision of the DNA sequence located between said first and saidsecond recombining sites occurs.
 18. The method according to claim 17,wherein said DNA sequence encodes a selectable marker.
 19. The methodaccording to claim 18, further comprising introducing a third constructinto a plant cell obtained from said regenerated plant.
 20. The methodaccording to claim 12, wherein each of said recombining sites areselected from the group consisting of Lox, FRT, and R.
 21. Therecombinant nucleic acid construct according to claim 12, furthercomprising regions of homology for integration into the plastid genome.22. A plant cell produced according to method of claim 12.